The protein turnover and trafficking of Chlamyopsin6 is regulated by IFT88 and IFT52 in the Chlamydomonas reinhardtii

Microbial rhodopsin-based optogenetics has been widely applied to diverse mammalian and plant cell types for controlling membrane potential mediated responses. However, trafficking of optogenetically active protein to the desired subcellular organelle is still a major concern in optogenetic field. This could be resolved by studying the trafficking mechanism of optogenetically active protein in the native system. Current study is focused on the trafficking of two of the microbial rhodopsins named Chalmyopsin5 and Chlamyopsin6 in a green alga, Chlamydomonas reinhardtii. Chlamyopsin5 and Chlamyopsin6 are modular in nature and possess rhodopsin, histidine kinase, response regulator and cyclase domain in tandem. Immunolocalization of Chlamyopsin5 and Chlamyopsin6 in wild strain suggests their different subcellular localization; Chlamyopsin5 in eyespot and Chlamyopsin6 in flagella. Extensive immunocytochemistry of Chlamyopsin5 and Chlamyopsin6 was performed in different intraflagellar transport (IFT) components-defective strains of Chlamydomonas to dissect their trafficking mode to the destined subcellular compartment. Our results indicated the trafficking of Chlamyopsin5 to the eyespot to be independent of IFT machinery while Chlamyopsin6 to the flagella to be IFT dependent. Further, we demonstrate that IFT88 and IFT52 stabilizes Chlamyopsin6 and IFT20 interacts with Chlamyopsin6 in Chlamydomonas. Protein interactome of Chlamyopsin5 and Chlamyopsin6 indicate their role in nitrogen assimilation, gametogenesis and photoprotection in co-ordination with other photoreceptors. Collectively, our study enabled us to understand the targeting of Chlamyopsins to the subcellular compartment (eyespot and flagella). Further research in this direction is required to resolve the current challenge of targeting of optogenetic protein to desired subcellular compartment. HighlightsO_LIChlamyopsin6 localizes in eyespot and flagella of Chlamydomonas reinhardtii and its trafficking is IFT-mediated. C_LIO_LIChlamyopsin5 localizes in the eyespot and its trafficking is independent of IFT machinery. C_LIO_LIDisruption of IFT52 and IFT88 results in reduced Chlamyopsin6 protein content within the cell. C_LIO_LIThe protein interactome suggests the crosstalk among photoreceptors to mediate light-triggered cellular responses. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/693822v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1041d79org.highwire.dtl.DTLVardef@12b06f1org.highwire.dtl.DTLVardef@1d4f970org.highwire.dtl.DTLVardef@1445a3d_HPS_FORMAT_FIGEXP M_FIG C_FIG